Lincomycin isothiouronium salts and process for preparing same

ABSTRACT

A peracylated trans-1-bromoglycoside is treated with thiourea in a tertiary amide, dipolar, aprotic solvent to form the corresponding peracylated cis- and trans-glycosyl isothiouronium salt, which is then converted to the corresponding peracylated 1-thioglycoses and alkyl peracylated 1-thioglycosides, and if desired, the cis anomers are isolated. 1-thioglycoses

United States Patent Bannister LINCOMYCIN ISOTHIOURONIUM SALTS ANDPROCESS FOR PREPARING SAME Inventor: Brian Bannister, Kalamazoo, Mich.

Assignee: The Upjohn Company, Kalamazoo,

Mich.

Filed: Nov. 3, 1969 Appl. No.: 873,716

US. Cl. ..260/2l0 R, 260/999 Int. Cl ..C07c 47/18 Field of Search..260/2l0 R References Cited UNITED STATES PATENTS l/ 1967 Bannister..260/2l0 R 3,366,624 l/l968 Argoudelis ..260/21O Primary ExaminerLewisGotts Assistant Examiner-Johnnie R. Brown Attorney-John Kekich and RomanSaliwanchik [57] ABSTRACT A peracylated trans-l-bromoglycoside istreated with thiourea in a tertiary amide, dipolar, aprotic solvent toform the corresponding peracylated cisand transglycosyl isothiouroniumsalt, which is then converted to the corresponding peracylatedl-thioglycoses and alkyl peracylated l-thioglycosides, and if desired,the cis anomers are isolated. l-thioglycoses 18 Claims, No DrawingsLINCOMYCIN ISOTHIOURONIUM SALTS AND PROCESS FOR PREPARING SAME BRIEFDESCRIPTION OF THE INVENTION This invention relates to a process forconverting peracylated transl -bromoglycoses to cisl thioglycosides andto novel compounds.

It is known that trans-l-thioglycoses, that is, lthioglycoses in whichthe l-thio group is in the trans position with respect to the 2-hydroxygroup, can be prepared by reacting a peracylated cisortrans-lbromoglycose with thiourea in acetone to form the correspondingisothiouronium salt, hydrolyzing this salt by mild alkaline hydrolysisto form the corresponding 1- thioglycose; and aklylating thisl-thioglycose and deacylating the resulting peracylated l-thioglycoside.This process yields trans-l-thioglycose isothiouronium salt, thetrans-l-thioglycose, and the alkyl trans-L thioglycoside.

In accordance with this invention, it has been found that when thereaction is carried out in a tertiary amide dipolar aprotic solvent,substantial quantities of the cisanomers are formed.

Thus this invention involves the reaction of a peracylatedtrans-l-bromoglycose with thiourea in a tertiary amide dipolar aproticsolvent, for example, hexamethyl phosphoric triamide, dimethylformamide,and N-methyl-Z-pyrrolidone, to form the corresponding 1- thioglycoseisothiouronium salt, hydrolizing this salt by mild basic hydrolysis tofree the corresponding 1- thioglycose, and concomitantly alkylating thelthioglycose thus formed to the corresponding alkyl 1- thioglycoside,and if desired, isolating the cis-anomer at any appropriate stage, andthereafter, if desired, removing the acyl group.

Peracylating refers to the replacement of all of the reactive hydrogenby a protective carboxacyl group, suitably with acetyl or other alkanoylgroups, for example, propionyl, butyryl, valeryl, hexanoyl, heptanoyl,octanoyl, and the isomeric forms thereof, or with benzoyl or likearalkanoyl groups.

Advantageously, the hydrolysis and alkylation are effected at the sametime as this minimizes decomposition of the l-thioglycose.Advantageously, also the hydrolysis and alkylation steps are conductedin the presence of an antioxidant, for example, sodium bisulfite, sodiumhydrosulfite (Na S O sodium pyrosulfite (sodium metabisulfite, Na S Oand the like, to minimize disulfide formation or to reduce any disulfideas it is formed.

A suitable alkylating agent is methyl iodide. Other alkylating agents,however, can be used, for example, alkyl halides, dimethyl sulfate,alkyl p-toluenesulfonate, and the like.

The invention may be more fully understood by reference to the followingexamples in which the solvent ratios are volume to volume and the partsare by weight unless otherwise specified. It is to be understood,however, that while the invention is thus exemplified with a particularl-bromo-B-glycose and the production of particular alkyla-thioglycosides, the invention is not thus limited but is applicable toany trans l-bromoglycose and to the production of the correspondingcis-alkyl thioglycoside.

EXAMPLEI Part A. 2-I-Iydroxyethyl N-acetyl-2',2,3,4-tetra-O-acetyl-7-O-methyl l-thio-a-lincosaminide 5 4 CH3 CHsO- AcNH-2'-Hydroxyethyl l-thio-a-celestosaminide (1.0 gm.) (Example 3 of US.Pat. No. 3,255,174) was left overnight in solution in pyridine (25 ccs.)and acetic anhydride l2 ccs. Removal of the solvent in vacuo gave acolorless oil which was dissolved in chloroform, washed with water,dilute aqueous hydrochloric acid, water, saturated aqueous sodiumbicarbonate, water and dried over anhydrous sodium sulfate. Solventremoval in vacuo gave a syrup (2.03 gms.) which on crystallization fromethyl acetate-Skellysolve B yielded 2'-hydroxyethylN-acetyl-2',2,3,4-tetra-O-acetyl-7-O- methyl-l-thio-oz-lincosaminide(Formula II) in squat, colorless prisms, m.p. l43-l44 C. Skellysolve Bis a brand of technical hexane.

Anal. CalCd. for C21I-I33O11NS:

C, 49.68;I-l,6.54;N, 2.76;S, 6.32%. Found:

C, 49.66; H, 6.50; N, 2.91; S, 6.34%. [0:1 ;+2l6(c,0.7746,Cl-ICl Part B.Methyl N-acetyl-2,3,4-tri-O-acetyl-7-O-methyll-thio-aand-B-lincosaminides 3 5 CH3 CH3 CHa0- CH:O

AcNH- AcNH- .AcO and AcO son, 4 f l LOH: l ll l Ac Ac A solution of 5.05gms. (1.62 ccs.) of bromine in 100 ccs. of chloroform was added overapproximately minutes from a pressure-equalized dropping funnel underanhydrous conditions to a stirred solution of 10 gms. of 2'-hydroxyethylN-acetyl-2',2,3,4-tetra-O- acetyl-l-thio-a-celestosaminide prepared bythe procedure of part A in 200 ccs. of chloroform. Initially, thebromine color disappeared immediately; later, a deep orange-red colordeveloped. After stirring for an additional 30 minutes at roomtemperature, solvent was removed on a rotating evaporator at C./7 mm.,giving a yellow-orange syrupy residue. This was redissolved inchloroform, the solvent removed in vacuo,

and the process repeated till the distillate became colorless, leaving ayellowish amorphous residue of lbromo-7-O-methyl-B-lincosaminetetraacetate of the formula AeNH-- x00 Br l 0A0 i 0A0 lll The residuewas dissolved in 200 cos. of dry dimethylformamide, 4.5 gms. of thioureawas added, and the reaction mixture (a colorless solution) stirredovernight at room temperature. There were thus EXAMPLE 2 0' Conversionof the trans-l-thioglycoside, methyl 6- acetamido-6-deoxy-2,3,4-tri-O-acetyll -thio-B-D- galactopyranoside to methyl6-amino-6-deoxy-l-thioa-D-galactopyranoside.

Without isolating these salts and after cooling in an icebath, 100 ccs.of water was added slowly, followed by 8.3 gms. of anhydrous potassiumcarbonate, 10.6 gms. of sodium bisulfite, and 28 gms. (12.3 ccs.) ofmethyl iodide. The mixture was stirred vigorously magnetically for 3hours, the cooling bath being removed after minutes.

Volatile materials were removed in vacuo at 40 C., and finally at 80 C./l mm. The yellowresidue was dissolved in a mixture of chloroform andwater, the aqueous layer extracted with chloroform, and the combinedchloroform extracts were washed twice with water and dried overanhydrous sodium sulfate. Removal of the solvent in vacuo gave acolorless amorphous residue (6.48 gms.). Thin-layer chromatography (lacetonezl Skellysolve B) showed a major zone of product with a smallzone of slightly higher R This material was chromatographed on silicagel (1.2 kilos, column dimensions 5.8 X 90 cms.) in the system 1acetone: 1 .5 Skellysolve B. After a 500 cc. forerun, 50 cc.fractionswere collected automatically, and elution of materials followedby thin-layer chromatography. Fractions nos. 145-173, inclusive,corresponded to the material of higher R nos. 185-310, inclusive,corresponded to the major product, and nos. 174-184, inclusive, were amixture of the two.

Removal of solvent in vacuo from combined fractions 145-173, inclusive,gave a colorless syrup (570 mgms.), which on crystallization from ethylacetate- Skellysolve B yielded methyl N-acetyl-2,3,4-tri-O-acetyl-7-O-methyl-l-thio-a-lincosaminide in small colorless prisms, m.p.212-213 C.

Anal. Calcd. for C H O NS:

C, 49.64; H, 6.71; N, 3.22; S, 7.36; MeO, 7.13%;

Found:

C, 49.72; H, 6.77; N, 3.36; S, 7.27; MeO, 7.08%.

[02], +229 (0, 0.7174, CHCl M. Wt: (Mass spec., M) 435.

Removal of solvent in vacuo from combined fractions 185-310, inclusive,gave slightly yellow amorphoussolid (4.23 gms.), which oncrystallization from ethyl acetate-Skellysolve B yielded methyl N-acetyl-2,3,4-tri-O-acety1-7-O-methyl-l-thio-B-lincosaminide in colorlessprisms, m.p. 187-l88 C.

Anal. Calcd. for c gHggogNsl C, 49.64; H, 6. 71; N, 3.22; S, 7.36; MeO,7.13%

Found:

C, 49.73; H, 6.95; N, 3.18; S, 7.64; MeO, 7.41%.

[01] +24 (c,.0.7484, CHCl M. Wt.: (Mass spec., M") 435.

Part A-2: 6-Acetamido-6-deoxy-BD-galactopyranosyl bromide methyl6-acetamido-6-deoxy-2,3,4-tri-0- acetyl-l-thio-B-D-galactopyranoside(21.59 gms., 1 mol.) was dissolved in chloroform (200 ccs.) in a 2 l.round-bottomed flask equipped with a magnetic stirring bar, apressure-equalized dropping funnel, and a drying tube, and a solution ofbromine (14.65 gms., 4.70 ccs., 1.6 mols.) in chloroform (200 ccs.)added slowly. After-an initial destruction of the color of the bromine,the solution assumed an orange-red hue. After an hour at roomtemperature beyond the addition, solvent was removed on a rotatingevaporator at 40 C./7 mm., the reddish-yellow syrupy residue redissolvedin chloroform, (as above), solvent removed in vacuo, and the processrepeated until the distillate was colorless.

Part B-2: Methyl, 6-acetamido-6-deoxy-2,3,4-tri-O- acetyll-thio-a-D-galactopyranoside Thiourea (13.1 gms., 3 mols.) andhexamethylphosphoric triamide (200 ccs.) were added, and the mixturestirred overnight at room temperature (drying tube). The reactionmixture then was cooled in an ice-bath, potassium carbonate (anhydrous,23.2 gms.) and sodium bisulfite (29 gms.) added, followed by water (200ccs.) slowly, the reaction mixture tending to become warm and toeffervesce. Methyl iodide (40 ccs., large excess) was then added slowly,again with effervescence, and the mixture stirred at room temperaturefor 3 hours.

All volatile solvent was removed on a rotating evaporator at 40 C./7mm., the residual solution dissolved in chloroform, washed twice withwater, and dried over anhydrous sodium sulfate. Solvent was removed on arotating evaporator at 40 C./7 mm., and then by distillation under highvacuum from an oil-bath at 1 10-120 C. The dark syrupy residue wasdissolved in chloroform, washed with water, dried over anhydrous sodiumsulfate, and the solvent removed at 40 C./ 7 mm.

The crude material was divided into two (16.4 and 16.5 gms.) andchromatographed separately on silica (3950 gms., column dimensions 10.5X 93 cm.) in the system 1 acetone:l.5 Skelly-solve B (technical hexane).Fractions (50 cc.) were collected automatically after a forerun of13.5 1. Fractions nos. 35 l-520, inclusive, were combined and yieldedthe oz-anomer (1.63 gms.); fractions 581-900, inclusive, gave theB-anomer (5.67 gms.), while fractions nos. 521-580, inclusive, consistedof a mixture (1.21 gms.) of the two anomers. The second column behavedvery similarly, and yielded 1.45 gms. of the a-anomer.

Fractions 521-900 were combined for recycling.

The a-anomer, methyl 6-acetamido-6-deoxy-2,3,4-tri-O-acetyl-1-thio-a-D-galactopyranoside, obtained initially as asyrup, crystallized readily from ethyl acetate-Skellysolve B incolorless, elongated platelets, m.p. l62l63 C.

Anal. Calcd. for C H O NS:

C, 47.73; H, 6.14; N, 3.71; S, 8.50%. M01. Wt.

377.41 Found:

C, 48.06; H, 6.50; N, 3.82; S, 8.69%.

[01],, +207 (c, 1.0006, chloroform).

Mol. Wt. (Mass Spec., M*) 377.

Part B-3 Conversion of methyl 6-acetamido-6-deoxy- 2 ,3 ,4-tri-O-acetyll-thio-a-D-galactopyranoside to methyl 6-amino-6-deoxy- 1-thio-a-D-galactopyranoside The a-thioglycoside tetracetate of Part B-2(3.08 gms.) was heated under gentle reflux with hydrazine hydrate (100ccs.) for 24 hours (oil-bath at 155 C.). Solvent was removed bydistillation from the colorless solution as completely as possible,finally under 7 mm. pressure giving a colorless syrup, which could notbe induced to crystallize. The mixture was chromatographed on silica gelin the system 1 methanol:l chloroform (v/v) till the faster movingcontaminants were removed: the column was then stripped with methanol.Solvent removal then gave methyl 6-amino-6-deoxy-l-thio-a-D-galactopyranoside as a colorless syrup 1.03 gms.).

This product is useful for the same purposes a m ethyl 6-amino-6-deoxy-1 -thio-a-D-galactooctopyranoside (a-MTL) as disclosed in U.S. Pat. No.3,380,992 and moreover can be acylated with trans-1-methyl-4-propyl-L-Z-pyrrolidinecarboxylic acid, and like acids, also asdisclosed in U.S. Pat. No. 3,380,992, to form 7,8-bisnorlincomycin andanalogs thereof useful as antibacterial agents.

The starting compound, 6-acetamido-6-deoxy-2,3,4-tri-O-acetyl-1-thio-B-D-galactopyranoside, was prepared as follows.6-Amino-6-deoxy-B-D-galactopyranose [Szarek et al., Can. J. of Chem.,43, 2345 (1965) and Saeki et al., Annual Reports of the Sankyo ResearchLaboratories, 19, 137 (1967) (from 18.0 gms. of6-amino-6-deoxy-l,2,3,4-di-O-isopropylidenea-D-galactopyranoside] wasdissolved in pyridine (100 cc.), acetic anhydride (50 cc.) added, thesolution left overnight at room temperature, and solvent removed ascompletely as possible on a rotating evaporator at 40 C./high vacuum.Thin layer chromatography of the resulting brown syrup (silica gel, 1acetonezl Skellysolve B) showed one spot only. The syrup was dissolvedin chloroform, washed with dilute hydrochloric acid (N), water,saturated aqueous sodium bicarbonate, water, and dried over anhydroussodium sulfate. Removal of the solvent in vacuo gave a pale yellowsyrup.

This pentaacetate (containing some a-anomer) was treated with a solutionof anhydrous hydrogen bromide -in glacial acetic acid (40 ccs.,saturated at 0 C.),

swirled by hand until all had dissolved and then left at roomtemperature for 3 hours. The tan, somewhat viscous reaction solution wasdiluted with chloroform (200 ccs.), and poured on ice. The chloroformlayer was removed, the aqueous layer extracted with chloroform, and thecombined extracts washed with cold water until the washings were neutralto Congo red indicating paper. The extracts were dried over anhydroussodium sulfate, and the solvent removed on a rotating evaporator at 40C./7 mm., to give an almost colorless syrup.

To the colorless syrup was added thiourea (5.01 gm., 3 mols.) andacetone ccs.) and the mixture heated under gentle reflux on thesteam-bath (Drierite tube) for l /2 hours, then left overnight at roomtemperature. To the reaction solution, cooled in an ice bath, was addedpotassium carbonate (anhydrous, 5.2 gms.), sodium bicarbonate (6.6gms.), water (50 ccs.) and methyl iodide (10 ccs., large excess) and themixture stirred vigorously magnetically for 2 hours.

Volatile solvent was removed on a rotating evaporator at 40 C./7 mm.,the aqueous residue extracted with chloroform, and the extracts washedwith water and dried over anhydrous sodium sulfate. Thin layerchromatography (silica gel, 1 acetonezl Skellysolve B) showed one zoneonly, of very slightly higher'Rf than the starting acetate, anddistinguished from it also in that it showed up rapidly on being sprayedwith periodate-permanganate reagent, which does not react with thepentaacetate. Removal of the solvent gave a colorless syrup (4.67 gms.).

Crystallization occurred from ethyl acetate-Skellysolve B to give themethyl 6acetamido-6-deoxy-2,3,4-tri-O-acetyl-1-thio-B-D-galactopyranoside as colorless prisms, m.p.116-117 C.

Anal. Calcd. for C H O NS:

C, 47.73; H, 6.14; N, 3.71; S, 8.50%. M01. Wt.

IL- u s Found:

C, 47.56; H, 6.1 1; N, 3.93; S, 8.69%.

[01],, +27 (c, 1.016, chloroform) Mol. Wt. (Mass Spec., M") 377.

1 claim:

1. In a process for converting a trans-glycosyl halide to thecorresponding isothiouronium halide by reaction with thiourea in asolvent, the improvement whereby substantial conversion tocis-isothiouronium halide is obtained characterized in that the solventis a tertiary amide dipolar aprotic solvent.

2. The process of claim 1 in which the solvent is hexamethylphosphorictriamide.

3. The process of claim 2 in which the solvent is dimethyl formamide.

4. The process of claim 1 in which the trans-glycosyl halide is6-acylamino-6-deoxy-2,3,4-tri-O-acyl-B-D- galactopyranosyl bromide orl-bromo-7-O-methyLB- lincosamine tetraacylate.

5. The process of claim 2 in which the trans-glycosyl galactopyranosylbromide or l-bromo-7-O-methyl-B- lincosamine tetraacylate.

6. The process of claim 3 in which the trans-glycosyl halide is6-acylamino-6-deoxy-2,3,4-tri-O-acyl-B-D- galactopyranosyl bromide orl-bromo-7-O-methyl-B- lincosamine tetraaacylate.

7. The processof claim 1 in which the cis-isothiouronium halide,-inadmixture with the trans-isothiouronium halide or separated therefrom,is hydrolyzed to the corresponding cis-l-thioglycose and concomitantlyalkylated to form an alkyl cis-l-thioglycoside.

8. The process of claim 7 in which the cis-isothiou- 10. The process ofclaim 9 in which the antioxidant is 5 a bisulfite.

11. The process of claim 9 in which the glycosyl isothiouronium halideis 6-acylamino-6-deoxy-2,3,4- tri-O-acyl-B-galactopyranosylisothiouroniumbromide or the l-isothiouronium bromide of7-O-methyl-B-lincosamine tetraacylate.

12. The process of claim 11 in which the antioxidant is a bisulfite.

13. A compound of the formula wherein Ac is lower alkanoyl or benzoyl.

14. A compound of claim 13 in which Ac is acetyl.

15. A compound of claim 13 in which the anomeric configuration is alpha.

16. A compound of claim 13 in which the anomeric configuration is beta.

17. A compound of claim 15 in which Ac is acetyl.

18. A compound of claim 16 in which Ac is acetyl.

2. The process of claim 1 in which the solvent is hexamethylphosphorictriamide.
 3. The process of claim 2 in which the solvent is dimethylformamide.
 4. The process of claim 1 in which the trans-glycosyl halideis 6-acylamino-6-deoxy-2,3,4-tri-O-acyl- Beta -D-galactopyranosylbromide or 1-bromo-7-O-methyl- Beta -lincosamine tetraacylate.
 5. Theprocess of claim 2 in which the trans-glycosyl halide is6-acylamino-6-deoxy-2,3,4-tri-O-acyl- Beta -D-galactopyranosyl bromideor 1-bromo-7-O-methyl- Beta -lincosamine tetraacylate.
 6. The process ofclaim 3 in which the trans-glycosyl halide is6-acylamino-6-deoxy-2,3,4-tri-O-acyl- Beta -D-galactopyranosyl bromideor 1-bromo-7-O-methyl- Beta -lincosamine tetraaacylate.
 7. The processof claim 1 in which the cis-isothiouronium halide, in admixture with thetrans-isothiouronium halide or separated therefrom, is hydrolyzed to thecorresponding cis-1-thioglycose and concomitantly alkylated to form analkyl cis-1-thioglycoside.
 8. The process of claim 7 in which thecis-isothioUronium halide is 6-acylamino-6-deoxy-2,3,4-tri-O-acyl- Beta-galactopyranosylisothiouronium bromide or the 1-isothiouronium bromideof 7-O-methyl- Beta -lincosamine tetraacylate.
 9. In a process forconverting glycosyl isothiouronium halides to 1-thioglycosides byhydrolysis to the corresponding 1-thioglycose and concomitant alkylationthe improvement which comprises carrying out the reaction in thepresence of an antioxidant thereby to minimize conversion of the1-thioglycose to the corresponding disulfide.
 10. The process of claim 9in which the antioxidant is a bisulfite.
 11. The process of claim 9 inwhich the glycosyl isothiouronium halide is6-acylamino-6-deoxy-2,3,4-tri-O-acyl- Beta -galactopyranosylisothiouronium bromide or the 1-isothiouronium bromide of 7-O-methyl-Beta -lincosamine tetraacylate.
 12. The process of claim 11 in which theantioxidant is a bisulfite.
 13. A compound of the formula
 14. A compoundof claim 13 in which Ac is acetyl.
 15. A compound of claim 13 in whichthe anomeric configuration is alpha.
 16. A compound of claim 13 in whichthe anomeric configuration is beta.
 17. A compound of claim 15 in whichAc is acetyl.
 18. A compound of claim 16 in which Ac is acetyl.